Throughout this application, various patents and publications are references and citations provided for them. The disclosure of these patents and publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains.
Much of the disclosure of U.S. Application Ser. No. 805,279 has also been published. See, e.g., European Patent Application No. 86116906.8, published Aug. 7, 1987.
Current DNA probe methodology basically involves attaching target DNA to a nitrocellulose filter by bringing it into contact with the filter directly or via the Southern transfer technique from an agarose gel. The DNA is then denatured and the filters baked to ensure firm attachment. Generally, the preparation of the DNA and the running of the gels is a time consuming, costly process requiring a reasonably high technical skill level.
The next step is to prepare the probe DNA. Probe DNA is prepared by labelling radioactively specific DNA by nick translation, polynucleotide kinase, or some other polymerase type copy reaction using nucleotides labelled with .sup.32 P. Once prepared, the probe DNA is permitted to hybridize with the bound target DNA. Hybridization is allowed to proceed at a suitable temperature, typically for several hours. The DNA probe will associate to form hybrid duplexes with any of the bound target DNA that has complementary base sequences. Extraneous material, including unbound probe DNA, is then washed away from the filter and the filter is then exposed to film sensitive to the radioactive label.
European Patent Application Publication No. 0 067 597, (Bender et al.) published Dec. 22, 1982 discloses oligonucleotides and a process for their preparation which comprises incorporating ribonucleotide units at specific locations in deoxyribonucleotide chains to provide predetermined cleavage sites which allow ease of chain cleavage. Although the products from their process are said to be useful for separating mixtures of nucleotide and polynucleotide products, Bender et al. do not teach a method for detecting target nucleic acid molecules based upon the amplification of probe fragments.
International Patent Application No. WO 84/03520 (Malcom et al.), published Sept. 13, 1984 discloses a method of detecting nucleic acid sequences which utilizes tandem hybridization of a nucleic acid probe and an enzyme containing marker. The method involves contacting the probe with a sample containing a complementary target sequence under hybridizing conditions. Before or after hybridization with the target sequence, the probe is attached by hybridization to an enzyme labelled marker polynucleotide which has a sequence complementary to a sequence on the probe.
U.S. Pat. No. 4,358,535 (Falkow et al.) discloses radioactively labeled nucleotide probes which are complementary to a target nucleic acid sequence of interest and a method of using these probes to detect the presence of a pathogen from which the target nucleic acid sequence is derived. The method comprises first fixing the target nucleic acid sequence to an inert support before hybridization with the probe. Next, the fixed nucleic acid is contacted with the radioactively labeled probe under hybridizing conditions, with hybridization taking place on the solid support. Then, the presence of the target nucleic acid sequence is determined by detecting the presence of any label on the inert support. A disadvantage of such a system is that the probe itself cannot be immobilized. If the probe of Falkow et al. is immobilized, rather than the target nucleic acid sequence, then the label molecules of the immobilized probe will be bound to the solid support regardless of whether the probe has hybridized with a target nucleic acid sequence. The result would not permit the detection of the presence of target nucleic acid.
European Patent Application Publication No. 0 117 440 discloses non-radioactive chemically labeled polynucleotide probes and methods of using the probes. The methods disclosed are similar to the method of Falkow et al. in that the target nucleic acid sequence is fixed to a solid support before hybridization.
Recently, other detection systems have been developed, such as fluorescent tags or color change enzyme systems. However, such systems have had significant problems with sensitivity and background levels (noise).
U.S. Pat. No. 4,362,867 (Paddock) discloses a hybrid nucleic acid construction which comprises two complementary deoxynucleotide sequences which are hybridized to form a double-stranded helical structure. Situated between and covalently bonded to the two deoxynucleotides is a ribonucleotide sequence. The construction forms a single unit, in which none of the nucleotide sequences repeat themselves.
U.S. Pat. No. 4,683,195 (Mullis, et al.) discloses a process for amplifying and detecting target nucleic acid sequences comprising treating separate complementary strands of the nucleic acid with a molar excess of two olignucleotide primers. By extending the primers to form complementary primer extension products which act as templates for synthesizing the desired nucleic acid sequence, the sequence so amplified is detected. A disadvantage of this process is the requirement of thermal cycling and the lack of constant temperature conditions.
In U.S. pat. No. 4,683,194 (Saiki et al.), a method is disclosed for detectiong the presence or absence of a specific site in a nucleic acid sequence using an oligonucleotide probe that is complementary to one strand of the nucleic acid sequence spanning the restriction site. Saiki et al. teach that their nucleic acid, i.e., oligonucleotide, probe, may be used as is or the sequence it contains can be amplified to increase sensitivity using the process disclosed in U.S. Pat. No. 4,683,202. Such an amplifying process suffers from the disadvantages inherent in U.S. Pat. No. 4,683,195, discussed above.